Oligonucleotides have been used in various biological and biochemical applications. Oligonucleotides have been used as primers and probes for the polymerase chain reaction (PCR), as antisense agents used in target validation, drug discovery and development, as ribozymes, as aptamers, and as general stimulators of the immune system. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients. More recently, Kynamro® (Mipomersen sodium injectable; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) has achieved marketing clearance (2013) from the U.S. Food and Drug Administration (FDA), and is currently a treatment of homozygous familial hypercholesterolemia (HoFH). The widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for their synthesis.
Synthesis of oligonucleotides is generally performed on solid support by the repeated coupling of phosphoramidite monomers until the predetermined length and sequence is achieved. The resulting full length oligonucleotide is then cleaved from the solid support and purified with a 5′-hydroxyl protecting group left on. The industry standard 5′-hydroxyl protecting group is the 4,4′-dimethoxytrityl (DMT) group. The phosphoramidite method is well known in the art (see for example: Beaucage and Caruthers (1981) Tetrahedron Letters 22:1859-1862; McBride and Caruthers (1983) Tetrahedron Letters 24:245-248; Sinha et al. (1984) Nucleic Acids Res. 12:4539-4557 and Beaucage and Iyer (1992) Tetrahedron 48:2223-2311, each of which is incorporated herein by reference in its entirety).
Large scale synthesis of oligomeric compounds using the phosphoramidite approach is generally performed using solid phase chemistries wherein oligomeric compounds are assembled in an iterative process on a solid support. A first monomer subunit is coupled to a free hydroxyl group attached to a solid support via a series of chemical reactions. This series of chemical reactions is repeated in an iterative manner for each additional monomer subunit until an oligomeric compound having a predetermined length and base sequence is synthesized. After the oligomeric compound has been cleaved from the solid support the DMT-on oligomeric compound is purified by reverse phase liquid chromatography. When the 5′-terminal protecting group is a 4,4′-dimethoxytrityl (DMT) group the oligomeric compound is referred to as a DMT-on oligomeric compound. The trityl group is normally left to simplify the purification step. In certain embodiments, the trityl group is removed before the cleavage of the oligomeric compound from the solid support.
Treatment of the DMT-on oligomeric compound with an acidified aqueous solution removes the 5′-trityl group. Neutralization of the acid quenches the reaction and the resulting detritylated oligomeric compound is precipitated using ethanol. One unwanted side reaction that occurs during this detritylation step is depurination. It has been observed experimentally that when standard methods are used (22° C., pH 3.5, 50 mg/g oligonucleotide concentration, see also examples 1 and 2), the rate of detritylation depends on the 5′-terminal bases especially the terminal base (A>G>T>C; see Example 3), and as the rate of detritylation decreases the percent of depurination increases. Oligomeric compounds having a C or a T at the 5′-end and several 2′-deoxy purine nucleosides have been shown to have longer detritylation times with the detritylated product having higher percentage of depurination. Consequently, there remains a long-felt need for improved methods for performing the final detritylation step that minimize depurination.
Detritylation under warm conditions with mildly acidic buffers to try to limit depurination has been reported (see Salon et al., Nucleosides, Nucleotides and Nucleic Acids, 2011, 30, 271-279).